Mycoplasma pneumoniae IgG ELISA

Transcription

1 Instructions for Use Mycoplasma pneumoniae IgG ELISA Enzyme immunoassays (microtiter strips) for the qualitative and quantitative determination of IgG antibodies against Mycoplasma pneumoniae in human serum and plasma RE x8 2-8 C I B L I N T E R N A T I O N A L G M B H Flughafenstrasse 52a Phone: +49 (0) IBL@IBL-International.com D Hamburg, Germany Fax: +49 (0)

2 1. INTENDED USE Enzyme immunoassay (microtiter strips) for the qualitative and quantitative determination of IgG antibodies against Mycoplasma pneumoniae in human serum and plasma. 2. SUMMARY AND EXPLANATION Mycoplasma pneumoniae is besides various viruses like rhinovirus, coronavirus, influenza, parainfluenza or adenovirus the reason for the classical common cold. Mycoplasms belong to the Mollicutes class. Common characteristics of the six eubacterial genera is the lack of a bacterial cell wall, osmotic fragility and small dimensions. The genome is significantly smaller compared to gram-positive and gram-negative bacteria. They depend on a host cell, respectively, on a host organism like a parasite. Mycoplasma pneumoniae is a human pathogenic bacterium causing tracheobronchitis and primary atypical pneumonia. Also secondary diseases like infarction, encephalitis, chronic neuropathy and the Guillain-Barre syndrome sometimes occur with a M. pneumoniae infection. In the laboratory, besides the cold agglutinin test and complement fixation reaction (CF), the ELISA is the method of choice showing an excellent sensitivity and the possibility to differentiate between the immunogloblin classes. Specific IgA antibodies are developed more regularly and more rapidly than IgM during an acute infection. IgA titres also decrease earlier than IgM or the late-peaking IgG response. Various studies showed that the determination of all the three immunoglobulin classes is necessary to monitor each step of the clinical course. 3. TEST PRINCIPLE Solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. The wells are coated with antigen. Specific antibodies of the sample binding to the antigen coated wells are detected by a secondary enzyme conjugated antibody (E-Ab) specific for human IgG. After the substrate reaction the intensity of the color developed is proportional to the amount of IgG-specific antibodies detected. Results of samples can be determined directly using the standard curve. 4. WARNINGS AND PRECAUTIONS 1. For in-vitro diagnostic use only. For professional use only. 2. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood. 3. In case of severe damage of the kit package please contact IBL or your supplier in written form, latest one week after receiving the kit. Do not use damaged components in test runs, but keep safe for complaint related issues. 4. Obey lot number and expiry date. Do not mix reagents of different lots. Do not use expired reagents. 5. Follow good laboratory practice and safety guidelines. Wear lab coats, disposable latex gloves and protective glasses where necessary. 6. Reagents of this kit containing hazardous material may cause eye and skin irritations. See MATERIALS SUPPLIED and labels for details. Material Safety Data Sheets for this product are available on the IBL- Homepage or upon request directly from IBL. 7. Chemicals and prepared or used reagents have to be treated as hazardous waste according to national biohazard and safety guidelines or regulations. 8. The cleaning staff should be guided by the professionals regarding potential hazards and handling. 9. Avoid contact with Stop solution. It may cause skin irritations and burns. 10. Some reagents contain sodium azide (NaN 3) as preservatives. In case of contact with eyes or skin, flush immediately with water. NaN 3 may react with lead and copper plumbing to form explosive metal azides. When disposing reagents, flush with a large volume of water to avoid azide build-up. 11. All reagents of this kit containing human serum or plasma have been tested and were found negative for anti-hiv I/II, HBsAg and anti-hcv. However, a presence of these or other infectious agents cannot be excluded absolutely. For this reason reagents should be treated as potential biohazards in use and for disposal. Version / 6

3 5. STORAGE AND STABILITY The kit is shipped at ambient temperature and should be stored at 2-8 C. Keep away from heat or direct sunlight. The storage and stability of specimens and prepared reagents is stated in the corresponding chapters. The unopened reagents are stable until the expiry date indicated. The Kit is stable up to 3 months after the first opening when the Microtiterplate is packed in a tightly closed bag, the bottles are closed with their screw caps and the kit is stored at 2-8 C. 6. SPECIMEN COLLECTION AND STORAGE Serum, Plasma (EDTA, Heparin) The usual precautions for venipuncture should be observed. It is important to preserve the chemical integrity of a blood specimen from the moment it is collected until it is assayed. Do not use grossly hemolytic, icteric or grossly lipemic specimens. Samples appearing turbid should be centrifuged before testing to remove any particulate material. Storage: 2-8 C -20 C Keep away from heat or direct sunlight. Stability: 7 days > 7 days Avoid repeated freeze-thaw cycles. 7. MATERIALS SUPPLIED Quantity Symbol Component 1 x 12 x 8 MTP 1 x 15 ml ENZCONJ IgG 1 x 4 x 2 ml CAL A-D 1 x 60 ml DILBUF 1 x 60 ml WASHBUF CONC 1 x 15 ml TMB SUBS 1 x 15 ml TMB STOP 2 x FOIL 1 x BAG Microtiter Plate Break apart strips. Coated with specific antigen. Enzyme Conjugate IgG Red colored. Ready to use. Contains: anti-human IgG, conjugated to peroxidase (rabbit), protein-containing buffer, 0.01 % Methylisothiazolinone, 0.01 % Bromonitrodioxane and 5 mg/l ProClin. Standard A-D 1; 10; 30; 100 U/mL. Ready to use. Standard A = Negative Control Standard B = Cut-Off Control Standard C = Weakly Positive Control Standard D = Positive Control Contains: IgG antibodies against Mycoplasma, PBS, 0.01 % Methylisothiazolinone and 0.01 % Bromonitrodioxane. Diluent Buffer Ready to use. Contains: PBS Buffer, BSA, < 0.1 % NaN3. Wash Buffer, Concentrate (10x) Contains: PBS Buffer, Tween 20. TMB Substrate Solution Ready to use. Contains: TMB. TMB Stop Solution Ready to use. 0.5 M H2SO4. Adhesive Foil For covering of Microtiter Plate during incubation. Plastic Bag Resealable. For dry storage of non-used strips. 8. MATERIALS REQUIRED BUT NOT SUPPLIED 1. Micropipettes (Multipette Eppendorf or similar devices, < 3 % CV). Volumes: 5; 50; 100; 500 µl 2. Calibrated measures 3. Tubes (1 ml) for sample dilution 4. 8-Channel Micropipettor with reagent reservoirs 5. Wash bottle, automated or semi-automated microtiter plate washing system 6. Microtiter plate reader capable of reading absorbance at 450 nm (reference wavelength nm) 7. Bidistilled or deionised water 8. Paper towels, pipette tips and timer Version / 6

4 9. PROCEDURE NOTES 1. Any improper handling of samples or modification of the test procedure may influence the results. The indicated pipetting volumes, incubation times, temperatures and pretreatment steps have to be performed strictly according to the instructions. Use calibrated pipettes and devices only. 2. Once the test has been started, all steps should be completed without interruption. Make sure that required reagents, materials and devices are prepared ready at the appropriate time. Allow all reagents and specimens to reach room temperature (18-25 C) and gently swirl each vial of liquid reagent and sample before use. Mix reagents without foaming. 3. Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips for each component and specimen. Do not interchange caps. Always cap not used vials. Do not reuse wells/tubes or reagents. 4. Use a pipetting scheme to verify an appropriate plate layout. 5. Incubation time affects results. All wells should be handled in the same order and time sequences. It is recommended to use an 8-channel Micropipettor for pipetting of solutions in all wells. 6. Microtiter plate washing is important. Improperly washed wells will give erroneous results. It is recommended to use a multichannel pipette or an automatic microtiter plate washing system. Do not allow the wells to dry between incubations. Do not scratch coated wells during rinsing and aspiration. Rinse and fill all reagents with care. While rinsing, check that all wells are filled precisely with Wash Buffer, and that there are no residues in the wells. 7. Humidity affects the coated wells/tubes. Do not open the pouch until it reaches room temperature. Unused wells/tubes should be returned immediately to the resealed pouch including the desiccant. 10. PRE-TEST SETUP INSTRUCTIONS Preparation of Components The contents of the kit for 96 determinations can be divided into 3 separate runs. The volumes stated below are for one run with 4 strips (32 determinations). Dilute / dissolve 20 ml Component Diluent Relation Remarks Storage Stability WASHBUF CONC 180 ml bidist. water 1:10 Warm up at 37 C to dissolve crystals, if necessary. Mix vigorously. 2-8 C 8 weeks Dilution of Samples Sample to be diluted with Relation Remarks Serum / Plasma generally DILBUF 1:101 e.g. 5 µl µl DILBUF Samples containing concentrations higher than the highest standard have to be diluted further. Version / 6

5 11. TEST PROCEDURE 1. Pipette 100 µl of each Standard and diluted sample into the respective wells of the Microtiter Plate. In the qualitative test only Standard B is used. 2. Cover plate with adhesive foil. Incubate 60 min at C. 3. Remove adhesive foil. Discard incubation solution. Wash plate 3 x with 300 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 4. Pipette 100 µl of Enzyme Conjugate into each well. 5. Cover plate with new adhesive foil. Incubate 30 min at C. 6. Remove adhesive foil. Discard incubation solution. Wash plate 3 x with 300 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 7. For adding of Substrate and Stop Solution use, if available, an 8-channel Micropipettor. Pipetting should be carried out in the same time intervals for Substrate and Stop Solution. Use positive displacement and avoid formation of air bubbles. 8. Pipette 100 µl of TMB Substrate Solution into each well. 9. Incubate 20 min at C in the dark (without adhesive foil). 10. Stop the substrate reaction by adding 100 µl of TMB Stop Solution into each well. Briefly mix contents by gently shaking the plate. Color changes from blue to yellow. 11. Measure optical density with a photometer at 450 nm (Reference-wavelength: nm) within 60 min after pipetting of the Stop Solution. 12. QUALITY CONTROL The test results are only valid if the test has been performed following the instructions. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or comparable standards /laws. User and/or laboratory must have a validated system to get diagnosis according to GLP. All standards/controls must be found within the acceptable ranges as stated on the QC Certificate. If the criteria are not met, the run is not valid and should be repeated. Each laboratory should use known samples as further controls. It is recommended to participate at appropriate quality assessment trials. In case of any deviation the following technical issues should be proven: Expiration dates of (prepared) reagents, storage conditions, pipettes, devices, incubation conditions and washing methods. 13. CALCULATION OF RESULTS The evaluation of the test can be performed either quantitatively or qualitatively Qualitative Evaluation The Cut-off value is given by the optical density (OD) of the Standard B (Cut-off standard). The Cut-off index (COI) is calculated from the mean optical densities of the sample and Cut-off value. If the optical density of the sample is within a range of 20% around the Cut-off value (grey zone), the sample has to be considered as borderline. Samples with higher ODs are positive, samples with lower ODs are negative. For a quantification, the Cut-off index (COI) of the samples can be formed as follows: COI = OD Sample OD Standard B Quantitative Evaluation The obtained OD of the standards (y-axis, linear) are plotted against their concentration (x-axis, logarithmic) either on semi-logarithmic graph paper or using an automated method. A good fit is provided with cubic spline or point-to-point curve, because these methods give the highest accuracy in the data calculation. For the calculation of the standard curve, apply each signal of the standards (one obvious outlier of duplicates might be omitted and the more plausible single value might be used). The concentration of the samples can be read directly from the standard curve. The initial dilution has been taken into consideration when reading the results from the graph. Results of samples of higher predilution have to be multiplied with the dilution factor. Version / 6

6 Samples showing concentrations above the highest standard have to be diluted as described in PRE-TEST SETUP INSTRUCTIONS and reassayed. Typical Calibration Curve (Example. Do not use for calculation!) Standard U/mL OD Mean A B C D INTERPRETATION OF RESULTS Method Range Interpretation < 8 U/mL negative Quantitative 8 12 U/mL equivocal (Standard curve) > 12 U/mL positive Qualitative (Cut-off Index, COI) < 0.8 negative equivocal > 1.2 positive The results themselves should not be the only reason for any therapeutical consequences. They have to be correlated to other clinical observations and diagnostic tests. 15. EXPECTED VALUES In an in-house study, apparently healthy subjects showed the following results: Interpretation Ig Isotype n positive equivocal negative IgG % 22.9 % 66.7 % 16. LIMITATIONS OF THE PROCEDURE Specimen collection and storage have a significant effect on the test results. See SPECIMEN COLLECTION AND STORAGE for details. Azide and thimerosal at concentrations > 0.1 % interfere in this assay and may lead to false results. The following blood components do not have a significant Hemoglobin 8.0 mg/ml effect (+/-20% of expected) on the test results up to the below Bilirubin 0.3 mg/ml stated concentrations: Triglyceride 5.0 mg/ml 17. PERFORMANCE Intra-Assay Precision 7.9 % Inter-Assay Precision 7.7 % Inter-Lot Precision % Analytical Sensitivity 1.12 U/mL Recovery % Linearity Range (U/mL) Serial dilution up to Range (%) Cross Reactivity : % No cross-reactivities were found to: RSV, Influenza, Parainfluenza, Adenovirus. Clinical specificity 80 % Clinical sensitivity 96 % Version / 6

7 18. PRODUCT LITERATURE REFERENCES 1. Ansari MHK, Rasmi Y, Manafi M, Rahimipour A, Ghadermarzi E, The Evaluation of Helicobacter pylori infection and cardiovascular disease risk factors with artheriosclerosis, Urmia Medical J 14(9): (2010) 2. Chaudhry R, Varshney AK, Malhotra P, Adhesion proteins of Mycoplasma pneumoniae, Front Biosci 1 (12): (2007) 3. Chia WK, Spence L, Dunkley L, Bradbury W, Development of urease conjugated enzyme-linked immunosorbent assays (ELISA) for the detection of IgM and IgG antibodies against Mycoplasma pneumoniae in human sera, Diagn Microbiol Infect Dis 11: (1988) 4. Clyde WA, Clinical overview of typical Mycoplasma pneumoniae infections, Clin Infect Dis 17: (1993) 5. Daxboeck F, Krause R, Wenisch C, Laboratory diagnosis of Mycoplasma pneumoniae infection, Clin Microbiol Infect 9(4): (2003) 6. Döller G, Döller PC, Jacobs E, Schuy W, Zur Differentialdiagnostik von Infektionen des Respirationstrakts, Diagnose & Labor 43: (1993) 7. Drasbek M, Nielsen PK, Persson K, Birkelund S, Christiansen G, Immune response to Mycoplasma pneumoniae P1 and P116 in patients with atypical pneumonia analyzed by ELISA, BMC Microbiol 4: 1-10 (2004) 8. Dumke R, Von Baum H, Lück PC, Jacobs E, Subtypes and variants of Mycoplasma pneumoniae: local and temporal changes in Germany and absence of a correlation between the genotype in the respiratory tract and the occurrence of genotype-specific antibodies in the sera of infected patients, Epidemiol Infect 138(12): (2010) 9. Ferwerda A, Moll HA, de Groot R, Respiratory tract infections by Mycoplasma pneumoniae in children: a review of diagnostic and therapeutic measures, Eur J Pediatr 160: (2001) 10. Granström M, Holme T, Sjögren AM, Örtqvist A, Kalin M: The role of IgA determination by ELISA in the early serodiagnosis of Mycoplasma pneumoniae infection, in relation to IgG and μ-capture IgM methods, Med Microbiol. 40: (1994) 11. Hammerschlag MR, Mycoplasma pneumoniae infections, Curr Opin Infect Dis 14: (2001) 12. Hirschberg L, Holme T, Krook A, Vikerfors T, IgG response to Mycoplasma pneumoniae in patients with communityacquired pneumonia determined by ELISA, APMIS 96: (1988) 13. Jacobs E, Mycoplasma pneumoniae: now in the focus of clinicians and epidemiologists, Eurosurveillance 17 (6): 1-3 (2012) 14. Seggev JS, Sedmak GV, Kurup VP, Isotype-specific antibody responses to acute Mycoplasma pneumoniae infection, J Ann Allergy Asthma Immunol 77: (1996) 15. Sillis M, The limitations of IgM assays in the serological diagnosis of Mycoplasma pneumoniae infections, J Med Microbiol 33: (1990) 16. Tuuminen T, Suni J, Kleemola M, Jacobs E, Improved sensitivity and specificity of enzyme immunoassays with P1-adhesin enriched antigen to detect acute Mycoplasma pneumoniae infection, J Microb Meth 44(1): (2001) Version / 6

8 Symbols / Symbole / Symbôles / Símbolos / Símbolos / Σύμβολα REF LOT CONC LYO IVD Cat.-No.: / Kat.-Nr.: / No.- Cat.: / Cat.-No.: / N.º Cat.: / N. Cat.: / Αριθμός-Κατ.: Lot-No.: / Chargen-Bez.: / No. Lot: / Lot-No.: / Lote N.º: / Lotto n.: / Αριθμός -Παραγωγή: Use by: / Verwendbar bis: / Utiliser à: / Usado por: / Usar até: / Da utilizzare entro: / Χρησιμοποιείται από: No. of Tests: / Kitgröße: / Nb. de Tests: / No. de Determ.: / N.º de Testes: / Quantità dei tests: / Αριθμός εξετάσεων: Concentrate / Konzentrat / Concentré / Concentrar / Concentrado / Concentrato / Συμπύκνωμα Lyophilized / Lyophilisat / Lyophilisé / Liofilizado / Liofilizado / Liofilizzato / Λυοφιλιασμένο In Vitro Diagnostic Medical Device. / In-vitro-Diagnostikum. / Appareil Médical pour Diagnostics In Vitro. / Dispositivo Médico para Diagnóstico In Vitro. / Equipamento Médico de Diagnóstico In Vitro. / Dispositivo Medico Diagnostico In vitro. / Ιατρική συσκευή για In-Vitro ιάγνωση. Evaluation kit. / Nur für Leistungsbewertungszwecke. / Kit pour évaluation. / Juego de Reactivos para Evaluació. / Kit de avaliação. / Kit di evaluazione. / Κιτ Αξιολόγησης. Read instructions before use. / Arbeitsanleitung lesen. / Lire la fiche technique avant emploi. / Lea las instrucciones antes de usar. / Ler as instruções antes de usar. / Leggere le istruzioni prima dell uso. / ιαβάστε τις οδηγίες πριν την χρήση. Keep away from heat or direct sun light. / Vor Hitze und direkter Sonneneinstrahlung schützen. / Garder à l abri de la chaleur et de toute exposition lumineuse. / Manténgase alejado del calor o la luz solar directa. / Manter longe do calor ou luz solar directa. / Non esporre ai raggi solari. / Να φυλάσσεται μακριά από θερμότητα και άμεση επαφή με το φως του ηλίου. Store at: / Lagern bei: / Stocker à: / Almacene a: / Armazenar a: / Conservare a: / Αποθήκευση στους: Manufacturer: / Hersteller: / Fabricant: / Productor: / Fabricante: / Fabbricante: / Παραγωγός: Caution! / Vorsicht! / Attention! / Precaución! / Cuidado! / Attenzione! / Προσοχή! Symbols of the kit components see MATERIALS SUPPLIED. Die Symbole der Komponenten sind im Kapitel KOMPONENTEN DES KITS beschrieben. Voir MATERIEL FOURNI pour les symbôles des composants du kit. Símbolos de los componentes del juego de reactivos, vea MATERIALES SUMINISTRADOS. Para símbolos dos componentes do kit ver MATERIAIS FORNECIDOS. Per i simboli dei componenti del kit si veda COMPONENTI DEL KIT. Για τα σύμβολα των συστατικών του κιτ συμβουλευτείτε το ΠΑΡΕΧΟΜΕΝΑ ΥΛΙΚΑ. COMPLAINTS: Complaints may be submitted initially written or vocal. Subsequently they need to be filed including the test performance and results in writing in case of analytical reasons. WARRANTY: The product is warranted to be free from material defects within the specific shelf life and to comply with product specifications delivered with the product. The product must be used according to the Intended use, all instructions given in the instructions for use and within the product specific shelf life. Any modification of the test procedure or exchange or mixing of components of different lots could negatively affect the results. These cases invalidate any claim for replacement. LIMITATION OF LIABILITY: IN ALL CIRCUMSTANCES THE EXTENT OF MANUFACTURER S LIABILITY IS LIMITED TO THE PURCHASE PRICE OF THE KIT(S) IN QUESTION. IN NO EVENT SHALL MANUFACTURER BE LIABLE FOR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING DAMAGES FOR LOST PROFITS, LOST SALES, INJURY TO PERSON OR PROPERTY OR ANY OTHER INCIDENTAL OR CONSEQUENTIAL LOSS. IBL International GmbH Flughafenstr. 52A, Hamburg, Germany Tel.: + 49 (0) Fax: IBL@IBL-International.com WEB: Symbols Version 4.5 /